Water insoluble derivatives of polyanionic polysaccharides

ABSTRACT

A water insoluble, biocompatible composition that is formed by a method which combines, in an aqueous mixture, a polyanionic polysaccharide, a nucleophile, and an activating agent, under conditions sufficient to form the composition. Also, a water insoluble, biocompatible composition that is formed by a method which combines, in an aqueous mixture, a polyanionic polysaccharide, a modifying compound, a nucleophile and an activating agent under conditions sufficient to form the composition.

This is a divisional of application Ser. No. 07/833,973, filed Feb. 11,1992; which is a continuation-in-part of application Ser. No.07/703,254, filed May 20, 1991, abandoned; which is acontinuation-in-part of application Ser. No. 07/543,163, filed Jun. 25,1990, now U.S. Pat. No. 5,017,229; which is a continuation-in-part ofapplication Ser. No. 07/100,104, filed Sep. 18, 1987, now U.S. Pat. No.4,937,270.

BACKGROUND OF THE INVENTION

The present invention relates to biocompatible films and gels formedfrom chemically modified polyanionic polysaccharides.

Hyaluronic acid ("HA") is a naturally occurring mucopolysaccharidefound, for example, in synovial fluid, in vitreous humor, in bloodvessel walls and umbilical cord, and in other connective tissues. Thepolysaccharide consists of alternating N-acetyl-D-glucosamine andD-glucuronic acid residues joined by alternating β 1-3 glucuronidic andβ 1-4 glucosaminidic bonds, so that the repeating unit is--(1→4)--β--D--GlcA--(1→3)--β--D--GlcNAc--. In water, hyaluronic aciddissolves to form a highly viscous fluid. The molecular weight ofhyaluronic acid isolated from natural sources generally falls within therange of 5×10⁴ up to 1×10⁷ daltons.

As used herein the term "HA" means hyaluronic acid and any of itshyaluronate salts, including, for example, sodium hyaluronate (thesodium salt), potassium hyaluronate, magnesium hyaluronate, and calciumhyaluronate.

HA, in chemically modified ("derivatized") form, is useful as a surgicalaid, to prevent adhesions or accretions of body tissues during thepost-operation period. The derivatized HA gel or film is injected orinserted into the locus between the tissues that are to be kept separateto inhibit their mutual adhesion. To be effective the gel must remain inplace and prevent tissue contact for a long enough time so that when thegel finally disperses and the tissues do come into contact, they will nolonger have a tendency to adhere.

Chemically modified HA can also be useful for controlled release drugdelivery. Balazs et al., 1986, U.S. Pat. No. 4,582,865, states that"cross-linked gels of HA can slow down the release of a low molecularweight substance dispersed therein but not covalently attached to thegel macromolecular matrix." R. V. Sparer et al., 1983, Chapter 6, pages107-119, in T. J. Roseman et al., Controlled Release Delivery Systems,Marcel Dekker, Inc., New York, describes sustained release ofchloramphenicol covalently attached to hyaluronic acid via esterlinkage, either directly or in an ester complex including an alaninebridge as an intermediate linking group.

I. Danishefsky et al., 1971, Carbohydrate Res., Vol. 16, pages 199-205,describes modifying a mucopolysaccharide by converting the carboxylgroups of the mucopolysaccharide into substituted amides by reacting themucopolysaccharide with an amino acid ester in the presence of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ("EDC") inaqueous solution. They reacted glycine methyl ester with a variety ofpolysaccharides, including HA. The resulting products are water soluble;that is, they rapidly disperse in water or in an aqueous environmentsuch as is encountered between body tissues.

Proposals for rendering HA compositions less water soluble includecross-linking the HA. R. V. Sparer et al., 1983, Chapter 6, pages107-119, in T. J. Roseman et al., Controlled Release Delivery Systems,Marcel Dekker, Inc., New York, describe modifying HA by attachingcysteine residues to the HA via amide bonds and then cross-linking thecysteine-modified HA by forming disulfide bonds between the attachedcysteine residues. The cysteine-modified HA was itself water soluble andbecame water insoluble only upon cross-linking by oxidation to thedisulfide form.

De Belder et al., PCT Publication No. WO 86/00912, describe aslowly-degradable gel, for preventing tissue adhesions followingsurgery, prepared by cross-linking a carboxyl-containing polysaccharidewith a bi- or polyfunctional epoxide. Other reactive bi- orpolyfunctional reagents that have been proposed for preparingcross-linked gels of HA having reduced water solubility include:1,2,3,4-diepoxybutane in alkaline medium at 50° C. (T.C. Laurent et al.,1964, Acta Chem. Scand., vol. 18, page 274); divinyl sulfone in alkalinemedium (E. A. Balasz et al., U.S. Pat. No. 4,582,865, (1986); and avariety of other reagents including formaldehyde, dimethylolurea,dimethylolethylene urea, ethylene oxide, a polyaziridine, and apolyisocyanate (E. A. Balasz et al., U.K. Patent Appl. No. 84 20 560(1984). T. Malson et al., 1986, PCT Publication No. WO 86/00079,describe preparing cross-linked gels of HA for use as a vitreous humorsubstitute by reacting HA with a bi- or polyfunctional cross-linkingreagent such as a di- or polyfunctional epoxide. T. Malson et al., 1986,EPO 0 193 510, describe preparing a shaped article by vacuum-drying orcompressing a cross-linked HA gel.

SUMMARY OF THE INVENTION

In one aspect, the invention features a method for making a waterinsoluble biocompatible composition, the method including combining, inan aqueous mixture, a polyanionic polysaccharide, an activating agent,and a nucleophile, under conditions sufficient to form the composition.

In preferred embodiments of this aspect of the invention, the activatingagent which are used include benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate,O-benzotriazole-1-yl-N,N,N',N'-tetramethlyluronium hexafluorophosphate,bromotris(dimethylamino)phosphonium hexafluorophosphate, or thecorresponding halide salts thereof.

The preferred concentration of polyanionic polysaccharide in thereaction is 0.0002-0.1M, more preferably 0.0005-0.02M. The preferred pHfor carrying out the reaction is 3.5 to 8.0. The preferred reagentstoichiometry is at least 0.1 molar equivalents of activating agent permolar equivalent of polyanionic polysaccharide.

Another aspect of the invention features a method for making a waterinsoluble biocompatible composition, the method including combining, inan aqueous mixture, a polyanionic polysaccharide, an activating agent, amodifying compound, and a nucleophile, under conditions sufficient toform the composition.

In preferred embodiments of this aspect of the invention, modifyingcompounds include, 1-hydroxy-benzotriazole hydrate,1-hydroxybenzotriazole monohydrate, N-hydroxysulfosuccinimide,N-hydroxysuccinimide, 4-nitrophenol, 2-nitrophenol, 4-nitrothiophenol,2-nitrothiophenol, pentachlorophenol, pentafluorophenol, imidazole,tetrazole, 4-dimethylaminopyridine or other related compounds. Theactivating agent is preferably a diimide, more preferably acarbodiimide, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide.

Also in preferred embodiments of the second aspect of the invention, thepreferred polyanionic polysaccharide is present in the reaction at aconcentration of 0.0002-0.1M, more preferably 0.005-0.02M. The preferredpH for carrying out the reaction is 3.5 to 8.0. The preferred reagentstoichiometry is at least 0.1 molar equivalents of activating agent permolar equivalent of polyanionic polysaccharide, and at least 1 molarequivalent of modifying compound per molar equivalent of activatingagent. Preferred polyanionic polysaccharides for use in the methods ofthe invention include hyaluronic acid (HA), carboxymethyl cellulose(CMC), carboxymethyl amylose (CMA), chondroitin-6-sulfate, dermatinsulfate, heparin, and heparin sulfate; HA, CMC, and CMA are particularlypreferred. It is also well understood that two or more polyanionicpolysaccharides may be employed in the methods of the invention.

Also in both aspects of the invention, preferred nucleophilic compoundswhich are capable of reacting with the activated polyanionicpolysaccharide include amino acid amides (preferably leucinamidehydrochloride), monofunctional amines (preferably 3-amino-1-propanol),amino acid esters (preferably a methyl ester or a butyl ester, includingt-butyl ester), amino alcohols, amino thiols, amino phenols, aminocathechols, amino acids, salts of amino acids, peptides, proteins andother ambident nucleophilic compounds in which only one electron richmoiety reacts as a nucleophile with the activated polyanionicpolysaccharide.

The term "aqueous mixture", as used herein, generally refers to asolution composed primarily of water, but which may also comprise asmuch as 1 part in 20 of a polar aprotic solvent. Preferred aproticsolvents include acetonitrile, dimethylformamide,hexamethylphosphoramide, dimethylacetamide, N-methylpyrrolidinone,1,4-dioxane, and acetone.

A "polyanionic polysaccharide" is a polysaccharide containing more thanone negatively charged group, e.g., carboxyl groups at pH values aboveabout pH 4.0.

The terms "mole or molar concentration(M)" of polyanionicpolysaccharides, as used herein, refer to moles of the repeatingmonomeric unit contained within the polymer.

A polyanionic polysaccharide is said to be "activated", as that term isused herein, when it is treated in an aqueous mixture in a manner thatrenders the carboxyl groups on the polyanionic polysaccharide vulnerableto nucleophilic attack; and an "activating agent" is a substance that,in an aqueous mixture including a polyanionic polysaccharide, causes thepolyanionic polysaccharide to become so activated.

A "modifying" compound is defined as a reagent which, in the presence ofan activated polyanionic polysaccharide, reacts with the activatedcarboxyl moiety of the polyanionic polysaccharide to form a newactivated species capable of reacting with a nucleophile.

The activated polyanionic polysaccharides which comprise the waterinsoluble compositions produced by the method of the invention may be inthe form of a gel, or in the form of fibers. Blends may also be preparedby mixing various amounts of two or more different activated-polyanionicpolysaccharides. Preferably, blends consist of activated-HA andactivated-CMC, or activated-HA and activated-CMA.

The compositions and blends of the invention may be provided in the formof an adhesion prevention composition, e.g., in the form of a film,foam, or composition suitable for incorporation in a syringe. They mayalso include a pharmaceutically active substance dispersed throughoutmaking them useful as a drug delivery system. Suitable substancesinclude proteins, growth factors, enzymes, drugs, biopolymers, andbiologically compatible synthetic polymers.

The term "film", as used herein, means a substance formed by compressinga gel or fibers, or by allowing or causing a gel or fibers to dehydrate.Any gel or fibers of the invention may be formed into such a film.

The term "foam", as used herein, means a substance formed by introducinggas bubbles into the gels or fibers of the invention.

A "biocompatible" substance, as the term is used herein, is one that hasno medically unacceptable toxic or injurious effects on biologicalfunction.

We have discovered that a gel, foam, or film produced by treating apolyanionic polysaccharide with a suitable activating agent, may be madehaving decreased water solubility, without the use of any separatelyadded bi- or polyfunctional cross-linking agent.

A "water soluble" gel, or film, as that term is used herein, is onewhich, formed by drying an aqueous solution of 1% weight/weight ("w/w")sodium hyaluronate in water, having dimensions 3 cm×3 cm×0.3 mm, whenplaced in a beaker of 50 ml of distilled water at 20° C. and allowed tostand without stirring, loses its structural integrity as a film after 3minutes, and becomes totally dispersed within 20 minutes. A "waterinsoluble" film of the invention, as that phrase and like terms are usedherein, formed using a 1% aqueous solution of a polyanionicpolysaccharide, modified according to the invention, having the samedimensions and similarly allowed to stand without stirring in a beakerof 50 ml of distilled water at 20° C., is structurally intact after 20minutes; the film boundaries and edges are still present after 24 hours,although the film is swollen.

Because the gels and films are water insoluble, they can be thoroughlywashed with water before use to remove unreacted substances.

Gels, foams, and films of the invention can also be prepared in coloredform, by including a dye or stain in the reaction mixture. Such coloredfilms and gels can be more easily seen when in place or duringplacement, making them easier to handle during surgical procedures thancolorless ones.

The films, gels, and foams of the invention retain their strength evenwhen hydrated. Because they adhere to biological tissues without theneed for sutures, they are useful as postoperative adhesion preventionmembranes. They can be applied to tissue even in the presence ofbleeding.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Lysine-Modified HA

The gels, foams, and films of the invention are made generally asfollows. HA is dissolved in water and the pH of the resulting aqueousmixture is adjusted downward; then the dissolved HA is activated byadmixing a suitable activating agent, and a suitable lysine ester isadmixed with the activated HA and allowed to stand until the desired gelhas formed. The activating agent and the ester can be admixed in anysequence.

The preferred method of making the lysine-modified gels and films of theinvention will now be described in more detail. As one skilled in theart will appreciate, gels and films of the invention can be made usingprotocols that are within the method of the invention yet are differentin particulars from those described here.

A sample of hyaluronic acid or a salt of hyaluronic acid, such as sodiumhyaluronate, is dissolved in water to make an aqueous mixture. HA fromany of a variety of sources can be used. As is well-known, HA can beextracted from animal tissues or harvested as a product of bacterialfermentation. Hyaluronic acid can be produced in commercial quantitiesby bioprocess technology, as described for example in PCT PublicationNo. WO 86/04355. Preferably the concentration of HA in this firstaqueous mixture is in the range between 0.4% and 2.5% weight/weight("w/w"). Subsequent reactions are slower and less effective atsignificantly lower concentrations, while significantly higherconcentrations are difficult to handle owing to their high viscosity.

The aqueous HA mixture should be acidic, preferably having a pH betweenpH 4.0 and pH 5.0, more preferably between pH 4.3 and pH 4.75. At lowerpH values the preferred activating agent, EDC, is unstable, and athigher values the reaction rate is diminished. Preferably hydrochloricacid is added to adjust the pH, although other known acids can be used.

Once the pH of the aqueous HA mixture has been adjusted, an activatingagent is admixed. Preferred activating agents include carbodiimides,most preferably EDC (in some references this substance is termed1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide or "DEC") or ETC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide).

Then a nucleophilic lysine ester is admixed to the aqueous HA-activatingagent mixture. Preferred esters include methyl, ethyl, or t-butylesters. The lysine can be in the form of di-lysine, tri-lysine, orpolylysine, or their hydrochloride salts.

The lysine ester and the activating agent may be admixed to the pHadjusted HA mixture in any sequence, either all at once or gradually.

If a colored product is desired, a solution of a dye or stain such asthe blue dye "Brilliant Blue R", also known as "Coomassie™ BrilliantBlue R-250", distributed as "Serva Blue" by Serva, can be admixed to thereaction mixture at this point. The resulting product has a blue colorthat can provide a good contrast to the color of body tissues, makingthe film or gel easy to see while it is handled during surgery and onceit is in place.

Once the reagents (and the stain or dye, if any) have been admixed, thereaction mixture can be simply allowed to stand for a time, or it can becontinually or occasionally stirred or agitated.

Upon admixing of the reagents the pH rises, and can be maintained at thedesired pH by addition of acid as the reaction proceeds. We have found,however, that films and gels with various desired physical propertiescan be obtained by simply allowing the pH to rise as the reactionproceeds. The mode of addition of the reagents, particularly the EDC andthe lysine ester, is not critical, but the ratios of these reagents tothe HA is important. We have found that the best results are obtainedwhen the ratio of HA:EDC:Lysine ester ranges from 1:2:1 to 1:4:10. Lowervalues typically result in weaker, less insoluble products, while highervalues typically result in stronger, more insoluble products.

Polyanionic Polysaccharide-Modified HA

Polyanionic polysaccharide-modified HA gels and films are preparedgenerally by mixing HA (as described above) with a polyanionicpolysaccharide and an activating agent to form a water-insolubleprecipitate. The precipitate can be cast into thin membranes useful forpostoperative adhesion prevention. It can also be colored as describedabove. To increase the strength of films cast from the precipitate, thefilms may be subjected to dehydrothermal treatment in which they areheated under vacuum (about 30 mm Hg) at approximately 105° C. for 24 hr.

The polysaccharide and HA can be mixed together, after which theactivating agent is added. Alternatively, the polysaccharide may bereacted with the activating agent, followed by addition of HA. A thirdoption is to combine the HA with the activating agent, followed byaddition of the polysaccharide. Preferred activating agents are asdescribed above and include the carbodiimides EDC and ETC. The reactionis preferably carried out at a pH between 4 and 5. The preferredpolysaccharide concentration ranges from 0.005 to 0.1M, and is morepreferably in the range 0.01 to 0.02M. The preferred molar ratio ofpolysaccharide to activating agent is at least 1:1, more preferablyabout 1:4.

Activated Polyanionic Polysaccharides

Polyanionic polysaccharide gels, films, and foams are prepared generallyby mixing at least one polyanionic polysaccharide (e.g., HA, CMC, CMA)with an activating agent to form a water-insoluble material. Preferredactivating agents include the carbodiimides, EDC and ETC. The reactionmay be carried out at a pH between 3.5 and 8, with optimal reactionconditions occurring between pH 4.7 and 5.1. The polysaccharidemolecular weight used in the reaction may range from 9.0×10⁴ to 3.0×10⁶daltons, but preferably is between 2.5×10⁵ to 1.0×10⁶ daltons. Thepreferred molar ratio of polysaccharide to activating agent is at least1:1, and more preferably about 1:4. The insoluble material formed bythis method may be in the form of a gel or in the form of fibers and canbe used directly for adhesion prevention or drug delivery, or can becast onto flat molds and either air dried or lyophilized to yield thinfilms or foams.

In addition, blends can be prepared by mixing various amounts ofdifferent unpurified or purified activated-polyanionic polysaccharides.These blends are made homogeneous by mixing with overhead stirrersand/or high shear mixers. Unreacted activating agent may be removed fromthe unpurified mixture by molecular weight sizing, dialysis,dialfiltration or fractional precipitation with a water-soluble solvent,according to standard methods, prior to use. The purified mixture can beused directly for adhesion prevention and/or drug delivery, or may becast onto flat molds and either air dried or lyophilized to form filmsor foams.

Bop reagent-activated Polyanionic Polysaccharides

Polyanionic polysaccharide water-insoluble gels, films, and foams arealso prepared generally by dissolving at least one polyanionicpolysaccharide (e.g. HA, CMC, CMA) in an aqueous mixture; activating thepolyanionic polysaccharide with an activating agent such asbenzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(Bop-reagent); and reacting the activated polyanionic polysaccharidewith a suitable nucleophile to form the desired insoluble composition.

The reaction may be carried out at a pH between 3.5 and 8, with optimalreaction conditions between pH 4.6 and 5.0. The molecular weight of thepolyanionic polysaccharide used in the reaction may range from 6.0×10²to 4×10⁶ daltons, but preferably greater than 5×10⁵ daltons. Thepreferred reagent stoichiometry is at least 0.1 molar equivalents ofactivating agent per molar equivalent of polyanionic polysaccharide, andat least 1 molar equivalent of nucleophide per molar equivalent ofpolyamionic polysaccharide.

One major unexpected advantage of the BOP activation of polyanionicpolysaccharide is that the molecular weight of the polyanionicpolysaccharide is not decreased upon coupling to the nucleophile. Thisresult is in contrast to reactions involving carbodiimides solely, inwhich we observe a decrease in HA molecular weight upon nucleophiliccoupling.

A non-obvious aspect of this invention is that organic solubleactivating agents, described previously for use only in organicsolvents, are able to effect, in an aqueous mileau, chemical coupling ofa nucleophile with the water-soluble polyanionic polysaccharides. Thisobservation presents a significant additional unexpected advantage inthat any unreacted activators can be removed from the water-insolubleproduct simply by extracting the reaction solution with any appropriatewater-immiscible, organic solvent. Examples of such solvents may includediethyl ether, methylene chloride, chloroform, ethyl acetate, ortetrahydrofuran.

Modified Carbodiimide-activated Polyanionic Polysaccharides

Polyanionic polysaccharide water-insoluble gels, films, and foams areprepared generally by dissolving at least one polyanionic polysaccharide(e.g., HA, CMC, CMA), in an aqueous mixture; activating the polyanionicpolysaccharide with an activating agent such as a diimide, e.g. EDC orETC; modifying the activated polyanionic polysaccharide with a modifyingcompound such as 1-hydroxybenzotriazole hydrate (HOBt),1-hydroxybenzotriazole monohydrate or one of the other compoundsdescribed above; and reacting the activated polyanionic polysaccharidewith a suitable nucleophile to form the desired insoluble composition.

The molecular weight range of the polyanionic polysaccharide used in thereaction may be 6.0×10² to 4.0×10⁶ daltons, but preferably is greaterthan 5.0×10⁵ daltons. The preferred reagent stoichiometry is at least0.1 molar equivalents of a activating compound per molar equivalent ofpolyanionic polysaccharide, and more preferably at least 1:1. Thepreferred reagent stoichiometry also encompasses at least one molarequivalent of modifying compound per molar equivalent of activatingagent. The reaction with the activating agent may be carried out at a pHbetween 3.5 and 8, with optimal reaction conditions occurring between4.0 and 4.8. The modification of the polyanionic polysaccharide with themodifying compound is carried out at a pH between 3.0 and 8.0, withoptimal modification conditions occurring between pH 3.3 and 4.5.

In the case where the activating agent used is a diimide, the modifyingcompound reacts with the intermediate O-acylisourea to form a new activecarbonyl group capable of being transferred to a nucleophile.

It is our discovery that organic soluble modifying reagents, whichdisplay low solubility in water, can effect the coupling of polyanionicpolysaccharides to nucleophiles in an aqueous mixture. A significantunexpected advantage of this solubility difference between modifier andpolyanionic polysaccharide is that the insoluble product can be purifiedby extraction with a suitable, water-immiscible organic solvent such aschloroform, methylene chloride, ethyl acetate, or diethyl ether, or byalcohol precipitation and trituration.

An additional unexpected advantage imparted by a coupling ofnucleophiles to a modified, activated polyanionic polysaccharide is thatthe reaction can be carried out under acidic conditions which providescontrol of the specificity in reactions with ambident nucleophiliccompounds (e.g., amino alcohols, amino acids, amino thiols, aminophenols, amino catechols, peptides, and proteins) so that only one ofpotentially several electron rich moieties are capable of reacting as anucleophile with the activated polyanionic polysaccharide.

Films and Gels

Polyanionic polysaccharides modified according to the above descriptionscan be cast as films in a straightforward manner. Typically the reactionmixture is poured into a vessel having the desired size and shape andallowed to air dry. In general films formed by drying mixtures pouredthickly, so that they have a lower surface area/volume, possess greaterstrength than films formed by drying thinner, higher surface area/volumemixtures.

Alternatively a film can be formed by compressing a gel under conditionsthat permit escape of water, as, for example, by compressing the gelbetween two surfaces, at least one of which is porous, as described, forexample, in EPO 0 193 510.

If desired, a gel or film can be washed prior to use by, for example,perfusion with water or 1M aqueous sodium chloride. Alternatively thereaction mixture can be dialyzed to remove residual reagents prior tocasting as a film. Washing to remove residual reagents orreagent-derived material such as substituted ureas is desirable if thefilm or gel is to be used for therapeutic applications. Gels or filmscolored blue with Brilliant Blue R as described above do not lose theircoloration during such washing. The removal of reagents or reactionproducts can be monitored by high pressure liquid chromatography.

DETAILED DESCRIPTION OF THE INVENTION

The invention is described in more detail in the following examples.These examples are given by way of illustration and are not intended tolimit the invention except as set forth in the claims.

EXAMPLE 1

In this example hydrogels were prepared using EDC as an activating agentand leucine methyl ester hydrochloride as a nucleophile.

Sodium hyaluronate (400 mg; 1.0 mmol of carboxyl groups) having amolecular weight between 1×10⁶ and 2×10⁶ was dissolved in 10 ml ofdistilled water. The pH of the aqueous solution was adjusted to pH 4.75by addition of 0.1N HCl. Then 314 mg of EDC (1.64 mmol) was added all atonce followed by 190 mg (1.05 mmol) of L-leucine methyl esterhydrochloride. The pH of the reaction mixture then rose to 6.2 over twohours. The reaction mixture was kept at room temperature for five hours,after which time it had formed a thick insoluble hydrogel. This hydrogelcould be washed with a 1M NaCl solution to remove residual reagentswithout loss of its physical properties.

EXAMPLE 2

In this example various EDC/leucine:HA ratios were used for comparisonof gel formation and properties.

The procedure was as in Example 1, using sodium hyaluronate (400 mg; 1.0mmol of carboxyl groups) in 15 ml of water. In separate experiments thefollowing quantities of EDC and leucine methyl ester hydrochloride werethen added: 153 mg EDC (0.8 mmol)/182 mg leucine methyl esterhydrochloride (1.0 mmol); 76 mg EDC (0.4 mmol)/90 mg leucine methylester hydrochloride (0.5 mmol); and 38 mg EDC (0.2 mmol)/45 mg leucinemethyl ester hydrochloride (0.25 mmol). Strong hydrogels were obtainedas in example 1 for the highest ratio of EDC and leucine methyl esterhydrochloride. At the lowest ratio of reactants (0.2 mmol/0.25 mmol to1.0 mmol HA carboxyl groups) a weak gel was obtained, which collapsed toa fluid after two weeks.

EXAMPLE 3

In this example the HA concentration was reduced by one-half forcomparison of resulting gel properties.

The procedure was as in example 1 except the HA (400 mg; 1.0 mmol ofcarboxyl groups) was dissolved in 30 ml of water rather than 15 ml(11/3% w/w HA). A hydrogel was formed, although it was weaker than thatobtained in Example 1.

EXAMPLE 4

In this example films were prepared using EDC as an activating agent andleucine methyl ester hydrochloride as a nucleophile.

Sodium hyaluronate (400 mg; 1.0 mmol of carboxyl groups) was dissolvedin 40 ml of distilled water. The pH of the solution was adjusted to pH4.75 by addition of 0.1N HCl. Then EDC (314 mg; 1.64 mmol) was added ina single portion, followed by 190 mg (1.05 mmol) of L-leucine methylester hydrochloride. The pH of the reaction mixture rose to 6.2 duringtwo hours, after which time the solution was poured into a petri dish ofarea 6360 mm², and allowed to dry to a film over a two day period. Filmsproduced in this manner were strong and insoluble in water and 1Maqueous NaCl. The films could be washed with water or aqueous NaCl as inExample 1 to remove residual reagents without loss of their physicalproperties. Infrared spectroscopic analysis of such films showed nocarbodiimide absorption at about 2130 cm⁻¹ and displayed absorptions atabout 1740 cm⁻¹, 1700 cm⁻¹, 1650 cm⁻¹, and 1550 cm⁻¹.

EXAMPLE 5

In this example various HA concentrations were used in making films forcomparison of resulting film properties.

The procedure described in example 4 was repeated, using three differentinitial HA concentrations made by dissolving the HA (400 mg; 1.0 mmol ofcarboxyl groups) in 30 ml, 40 ml, or 100 ml of distilled water. Filmsproduced using each of these initial concentrations of HA were strongand insoluble in water and 1M aqueous NaCl, showing that a range ofconcentrations of HA can be used. Each of these films could be washedwith water or aqueous NaCl without loss of its physical properties.

EXAMPLE 6

This example illustrates the effect of dialyzing the reaction mixtureprior to casting to form a film, as compared with washing the film afterforming it.

Sodium hyaluronate (400 mg in 40 ml of water), EDC (314 mg; 1.64 mmol)and L-leucine methyl ester hydrochloride (190 mg; 1.05 mmol) wereallowed to react as in Example 4. Upon completion of reaction (2 hours)the reaction mixture was dialyzed against water, through 12,000 NMWcutoff dialysis tubing in order to remove residual reagents. Thedialyzed mixture was then cast as a film as in Example 4. The film soobtained was strong and insoluble in water or 1M aqueous NaCl.

EXAMPLE 7

In this example films were formed by drying more thickly poured reactionmixtures, to compare the properties of films produced from dryingmixtures at differing surface area/volume.

A reaction mixture obtained as in Example 4 (40 ml reaction volume) wascast into a small petri dish (area 3330 mm ²). The film so obtained wasinsoluble in 1M aqueous NaCl and in water (100° C.; 1 hour).

EXAMPLE 8

In this example films were prepared using other amino acid esters and HAactivated with EDC.

A solution of HA (400 mg in 40 ml of H₂ O) was brought to pH 4.7 using0.1N HCl. Then EDC (314 mg; 1.6 mmol) was added all at once followed by1 mmol of the amino acid derivative. The reaction mixture was pouredinto a petri dish and allowed to dry. Insoluble films were obtained fromL-valine methyl ester hydrochloride, L-isoleucine methyl esterhydrochloride, L-proline methyl ester hydrochloride, and L-phenylalaninemethyl ester hydrochloride.

EXAMPLE 9

In this example films were prepared using a simple primary amine(aniline) as a nucleophile.

A solution of HA (400 mg in 40 ml of H₂ O) was brought to pH 4.7 using0.1N HCl. Then EDC (314 mg; 1.6 mmol) was added all at once followed by1 mmol of aniline. The reaction mixture was poured into a petri dish andallowed to dry, and insoluble films were obtained.

EXAMPLE 10

In this example films were prepared using other esters of leucine.

A solution of HA (400 mg in 40 ml of H₂ O) was brought to pH 4.7 using0.1N HCl. Then EDC (314 mg; 1.6 mmol) was added all at once followed by1 mmol of the leucine ester. The reaction mixture was poured into apetri dish and allowed to dry. Insoluble films were obtained from bothL-leucine ethyl ester hydrochloride and L-leucine t-butyl esterhydrochloride.

EXAMPLE 11

In this example gels were prepared using other amino acid methyl esters.

A solution of HA (400 mg in 15 ml of H₂ O) was brought to pH 4.7 and EDC(314 mg; 1.6 mmol) was added, followed by the amino acid derivative (1mmol). The reaction mixture formed a thick gel within from 5 to 24hours. Water insoluble gels were obtained using L-valine methyl esterhydrochloride, L-isoleucine methyl ester hydrochloride, L-argininemethyl ester hydrochloride, L-proline methyl ester hydrochloride, andL-histidine methyl ester hydrochloride.

EXAMPLE 12

In this example films were prepared using an amino acid amide(leucinamide) as a nucleophile.

A solution of HA (400 mg in 40 ml of H₂ O) was brought to pH 4.7 using0.1N HCl. Then EDC (314 mg; 1.6 mmol) was added all at once followed by1 mmol of L-leucinamide hydrochloride. The reaction mixture was pouredinto a petri dish and allowed to dry. and insoluble films were obtained.

EXAMPLE 13

In this example gels were prepared using leucine ethyl esterhydrochloride.

A solution of HA (400 mg in 15 ml of H₂ O) was brought to pH 4.7 and EDC(314 mg; 1.6 mmol) was added, followed by leucine ethyl esterhydrochloride (1.0 mmol). The mixture formed a thick, water insolublegel within from 5 to 24 hours.

EXAMPLE 14

In this example films and gels were prepared using ETC as the HAactivating agent.

Sodium hyaluronate (400 mg, 1.0 mmol of carboxyl groups) having amolecular weight in the range between 1×10⁶ and 2×10⁶ daltons wasdissolved in water (10 ml and 30 ml). The pH of each aqueous solutionwas adjusted to pH 4.75 by addition of 0.1N HCl. Then 475 mg of ETC (1.6mmol) was added all at once, followed by 190 mg (1.05 mmol) of L-leucinemethyl ester hydrochloride. The pH of this reaction mixture rose to pH6.2 over the next 2 hours. The reaction mixture containing 10 ml ofwater formed an insoluble gel. The reaction mixture containing 30 ml ofwater gave an insoluble film after drying.

EXAMPLE 15

This example illustrates the preparation of a colored film.

A solution of HA (400 mg in 30 ml of H₂ O) was brought to pH 4.75 as inexample 13 and then ETC (475 mg; 1.6 mmol) and leucine methyl esterhydrochloride (190 mg; 1.05 mmol) were added. A dilute solution of"Serva Blue" (5 mg/ml) dye in H₂ O (0.5 ml) was then added to thereaction mixture. The resulting mixture was poured into a Petri dish anda water insoluble blue film was obtained after 16 hours. The blue colorwas retained by the film when the film was washed with 1M NaCl and thenwith H₂ O.

EXAMPLE 16

This example illustrates the tissue biocompatibility of a film ofchemically modified HA.

Four strips of films prepared according to the procedure described inExample 4, and two USP negative control strips were surgically implantedinto the paravertebral muscle of White New Zealand rabbits (two pertest). The test sites were evaluated either macroscopically after 72hours or with complete histopathology after 7 days. In accordance withthe USP XXI, p. 1237, the test material met the requirements of the USPImplantation Test for the Evaluation of Plastic Materials.

EXAMPLE 17

This example illustrates the preparation of lysine-modified HA.

A 0.4%(w/w) solution of HA in water was prepared. The pH of thissolution was adjusted to between 4.3 and 4.75 by addition of acid. Toeach 100 ml of this solution was added 0.76 g of EDC with stirring untilthe EDC had completely dissolved. To each 100 ml of the HA/EDC solutionwas added 0.20 g of lysine methyl ester (LME) with stirring until theLME had completely dissolved. The addition of HA, EDC, and LME wasconducted at room temperature; once the final HA/EDC/LME solution hadbeen formed, it was stored at 4° C. until needed.

The LME-modified HA material can be processed into various shapes,sizes, and consistencies depending on the end application. If a thinsheet of the material is desired, the mixture can be poured onto a flatsurface. This material can then be turned into a solid by allowing thewater to evaporate under ambient or elevated temperatures. Analternative method of producing sheets of the material is to subject itto freeze drying. The pore size of the final product can be controlledby adjusting the initial freezing temperature. Curved surfaces and othershapes can be produced in a similar manner by initially casting the gelonto a negative image surface and then processing as described. Thedried sheet can be processed further, if desired, by pressing to adefined thickness in a Carver laboratory press. This is particularlyuseful for applications requiring placing a thin film between anatomicalstructures where space is limited.

Mechanical testing of the freeze-dried material, rehydrated in normalsaline, resulted in force to break values of 170-900 g/cm. Theelongation to break values for this material were between 33 and 62%.

EXAMPLE 18

This example illustrates the preparation of CMC-modified HA.

HA (0.4% w/w, 0.01M) and Aqualon-type CMC having a molecular weight of250,000 and a degree of substitution in the range 0.65 to 0.90 (0.19%w/w, 0.01M) were mixed together in aqueous solution at room temperature.The pH of the mixture was adjusted to and maintained at pH 4.7-4.8 byaddition of 1M HCl. To each 100 ml of this solution was added 0.67 g(0.04M) EDC. During reaction with EDC, the pH of the solution wasmaintained at pH 4.7-4.8 by addition of 0.1M HCl and the reactionallowed to proceed for 1 hour, during which time a precipitate formed.The unreacted EDC was removed from the precipitate by dialysis againstacidified water (pH 4.0) for 24 hours with 2 dialysate changes at 3 and19 hours. The HA/CMC slurry was then cast into flat molds and air driedfor 24 hours at room temperature.

HA/CMC membranes were shown to reduce the incidence of postoperativeadhesion formation in experimental animal models. In experiments usingthe rat cecal abrasion model, HA/CMC membranes were placed aroundsurgically abraded rat ceca; previous studies had demonstrated thatadhesions readily formed to the ceca of rats which had been abraded incontrolled fashion. Cecal adhesions in animal groups that receivedeither HA/CMC membranes or ORC membranes (Interceed TC7 membranesmarketed by Johnson & Johnson for adhesion prevention) were compared toadhesion controls in animals whose ceca were abraded but did not receiveany membrane. The results of these experiments showed that the HA/CMCmembranes consistently reduced adhesion formation compared to controlanimals and to animals that received the Interceed TC7 film.

EXAMPLE 19

This example illustrates the preparation of EDC-activated HA.

HA (1.0×10⁶ daltons) was dissolved in water to make a 0.8% w/v solutionby stirring overnight at 25° C. The pH of the reaction mixture wasadjusted to pH 4.75 with 0.1N HCl. EDC (4:1 molar ratio of EDC to HA,1.53% w/v final concentration) was added to this solution withcontinuous stirring and was maintained at a constant pH (4.7-5.1) forone hour by adding additional 0.1N HCl. Removal of the unreacted EDC andother low molecular weight impurities was performed by either molecularweight sizing, dialysis, or dialfiltration using standard methods. Awater-insoluble, clear gel was obtained after this process.

EXAMPLE 20

This example illustrates the effect of fractional precipitation ofEDC-activated HA with a water soluble solvent.

The procedure described in example 19 was repeated with the exceptionthat unreacted EDC and other low molecular weight impurities wereremoved by fractional precipitation using a suitable water-solublesolvent (e.g., C₁ -C₃ alcohols, acetone). Under these conditions, waterinsoluble fibers were produced.

EXAMPLE 21

This example illustrates the preparation of EDC-activated CMC.

CMC (250×10³ daltons) was dissolved in water to make a 0.8% w/v solutionby stirring at room ambient temperature (22°-25° C.) overnight. The pHof the reaction mixture was adjusted to pH 4.75 with 0.1N HCl. EDC (4:1molar ratio of EDC to CMC, 1.53% w/v final concentration) was added tothis solution with constant stirring and the pH was maintained between4.70 and 5.10 for one hour by adding additional 0.1N HCL. Removal of theunreacted EDC and other low molecular weight impurities was performed byeither molecular weight seizing chromatography, dialysis,dialfiltration, or fractional precipitation of the CMC with a suitablewater-soluble solvent (e.g., C₁ -C₃ alcohols, acetone). Water insolublefibers, approximately 300-800 μm long and 10-20 μm wide, are producedfrom these reaction conditions.

EXAMPLE 22

This example illustrates the preparation of a blend of EDC-activated HAwith EDC-activated CMC.

EDC-activated HA and CMC were prepared separately as described inExamples 19 and 21 but each reaction product was not purified prior toblending. Three hundred ml of the activated HA and 300 ml of theactivated CMC were placed in a 1000 ml beaker and blended with a Turraxbrand blender at 6000 rpm for 10 minutes at 25° C. This resulted mixturewas purified by dialysis against pH 4.0 water for 24 hours at a 20:1ratio with 3 dialysate exchanges. After dialysis the mixture was pouredinto a flat mold and air dried to a thin water insoluble film. Thequantity of fibers in the mixture can be controlled by varying therelative amount of activated CMC and activated HA that are blendedtogether.

EXAMPLE 23

This example illustrates the coupling of HA with histidine using HOBtand EDC.

To a solution of sodium hyaluronate (200 mg, 0.5 mmoles, MW 1,700,000)and 1-histidine (155.2 mg, 1.0 mmole) in water (40 mL) was added1-hydroxybenzotriazole hydrate (HOBt) (67.6 mg, 0.5 mmoles) followed byadjustment of the pH to 3.35 with 1N HCl. After stirring for one hour,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (154.4mg, 0.8 mmoles) was added and the pH was maintained between 4.0 and 4.8,by the addition of 1N HCl, for two hours. Saturated sodium carbonate wasadded to adjust the pH to 7.01 and the product was precipitated by theaddition of 95% ethanol (≈150 mL). The resulting solid was collected byvacuum filtration, washed with absolute ethanol (3×20 mL), and dried bylyophilization. The overall yield of modified polymer was 65%. Thematerial was used directly without further purification.

EXAMPLE 24

This example illustrates the coupling of HA with3-dimethylaminopropylamine using HOBt and EDC.

3-Dimethylaminopropylamine (102 mg, 1.0 mmoles) in water (3 mL) wasadjusted to pH 7.0 with 1N HCl and added to a solution of sodiumhyaluronate (200 mg, 0.5 mmoles, MW 1,7000,000) in water (40 mL).1-Hydroxybenzotriazole hydrate (HOBt) (67.6 mg, 0.5 mmoles) was addedand the pH adjusted to 3.35 with 1N HCl. After stirring for one hour,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (154.4mg, 0.8 mmoles) was added and the pH maintained at 4.6 for 50 min. bythe addition of 1N HCl and/or 10% w/v sodium carbonate. Saturated sodiumcarbonate was then added to adjust the pH to 7.0 and half of the productwas purified by either the ultrafiltration or ethanol precipitationmethods listed below.

An aliquot (20 mL) of the reaction solution was purified byultrafiltration (1,000 MWCO) at ambient temperature against three volumeexchanges with water. The retentate was concentrated to about 12 mL andused without further purification.

Another aliquot (20 mL) of the reaction solution was added to 10% w/vaqueous NaCl (5 mL) followed by the addition of 95% ethanol (≈75 mL).This resulted in the formation of a precipitate which was collected byvacuum filtration, washed with absolute ethanol (3×20 mL), and dried bylyophilization. The product was used directly to prepare a hydrogelwithout further purification.

EXAMPLE 25

This example illustrates the coupling of HA with dihydroxyphenylamine(di-DOPA) using HOBt and EDC.

To a solution of sodium hyaluronate (200 mg, 0.5 mmoles, MW 2,100,000)and d,1-DOPA (200 mg, 1.0 mmole) in water (40 mL) was addedl-hydroxy-benzotriazole hydrate (HOBt) (67.6 mg, 0.5 mmoles) followed byadjustment of the pH to 4.35 with 1N HCl. After stirring for one hour,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (154.4mg, 0.8 mmoles) was added and the pH was maintained between 4.0 and 4.8for two hours by the appropriate addition of either 1N HCl or 10% w/vaqueous sodium carbonate. Saturated sodium carbonate was added to adjustthe pH to 7.0 and the product was precipitated by the addition of 95%ethanol (≈150 mL). The resulting solid was collected by vacuumfiltration, washed with absolute ethanol (3×20 mL), and dried bylyophilization. The material was used directly to prepare the hydrogelwithout further purification. Care should be taken to limit the gelsexposure of the gel to air in order to protect the material fromoxidation.

EXAMPLE 26

This example illustrates the coupling of CMC with3-dimethylaminopropylamine using HOBt and EDC.3-Dimethlyaminopropylamine (102 mg, 1.0 mmoles) in water (3 mL) wasadjusted to pH 7.0 with 1N HCl and added to a solution of CMC (125 mg,0.5 mmoles, MW 250,000) in water (20 mL). 1-Hydroxybenzotriazole hydrate(HOBt) (67.6 mg, 0.5 mmoles) was added and the pH adjusted to 4.0 with1N HCl. After stirring for one hour,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (154.4mg, 0.8 mmoles) was added and the pH was maintained at 4.7 for one hourby the addition of 1N HCl and/or 10% sodium carbonate. At the end of thereaction, 10% sodium carbonate was added to adjust the pH to 7.0. Theproduct was obtained as a white solid after ethanol precipitation.

EXAMPLE 27

This example illustrates the coupling of Bop-activated HA with glycinemethyl ester.

To a solution of sodium hyaluronate (200 mg, 0.5 mmoles, MW 2,100,000)in water (40 mL) was addedbenzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(Bop) (442.3 mg, 1.0 mmoles) in dimethy-formamide (1 mL) followed by pHadjustment to 4.6 with 1N HCl. After stirring for 15 min., glycinemethyl ester hydrochloride (126 mg, 1.0 mmoles) was added and thereaction mixture was allowed to stir at ambient temperature for 40 hr.The reaction mixture was transferred to a separatory funnel andextracted with methylene chloride (3×50 mL). The aqueous layer wasremoved and the product was precipitated with 95% ethanol and collectedby vacuum filtration. The solid was washed with a small quantity ofabsolute ethanol and air dried. This material was used to prepare thehydrogel directly without further purification.

EXAMPLE 28

This example illustrates the coupling of Bop-activated HA with3-amino-1-propanol.

To a sodium hyaluronate (200 mg, 0.5 mmoles, MW 1,700,000) solution inwater (20 mL) was addedbenzotriazole-1-yloxytris(dimethylamino)phosphonium-hexafluorophosphate(442.3 mg, 1.0 mmoles). After 15 min, 3-amino-l-propanol (45.1 mg, 0.6mmoles) was added with dimethylformamide (3 mL) and the pH of thereaction mixture was adjusted to 4.8 with 1.0N HCl. After stirringovernight at ambient temperature, the pH of this reaction mixture wasadjusted to ≈7.0 with saturated sodium carbonate and the reactionmixture was extracted with methylene chloride (3×50 mL). The aqueousphase was separated and 95% ethanol (≈100 mL) was added to precipitatethe product. The solid was collected by vacuum filtration, washed withabsolute ethanol (3×20 mL), an dried under reduced pressure with a yieldof 60%. The material was used directly to prepare the hydrogel withoutfurther purification.

EXAMPLE 29

This example illustrates the formation of a hydrogel using the reactionsproducts of examples 23 through 28.

A solid portion of any of the reaction products of examples 23 through28 (20 mg) was added to a 0.9% w/v sodium chloride solution (20 mL), andthe mixture was allowed to stand overnight at ambient temperature. Afterthis time period, a clear, colorless hydrogel formed which was isolatedby decanting the remaining sodium chloride solution. These resultinggels then were used directly for physical and in vivo evaluations.

EXAMPLE 30

This example illustrates the coupling of HA and CMC with3-dimethylaminopropylamine using HOBt and EDC.

To a solution of sodium hyaluronate (4.0 g, 10 mmoles, MW 2,300,000) andcarboxymethyl cellulose (5.1 g, 20 mmoles, MW 250,000) in water (500 mL)was added 1-hydroxybenzotriazole hydrate (4.1 g, 30 mmoles) and3-dimethylaminopropylamine hydrochloride (4.2 g, 30 mmoles) followed bypH adjustment to 4.60 with 1N HCl. After all the chemicals dissolved,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (8.0 g, 40mmoles) was added and the pH maintained between 4.6 and 5.0 by theaddition of either 1N HCl, or 10% Na₂ CO₃, for 1 hour. The pH was thenadjusted to 5.0 with 10% Na₂ CO₃ and half of the solution was ethanolprecipitated with 4× the reaction volume of ethanol, washed withadditional ethanol, and air dried. The remaining half was dialyzed(12,000NMW cutoff dialysis tubing) against water adjusted to pH 4.75(20× volumes) for 24 hrs.

Use

The films, foams, or gels of the invention can be used as a surgicalaid, to prevent adhesions or accretions of body tissues during apost-operation or healing period, following procedures known in thesurgical arts, as described, for example, in DeBelder et al., PCTPublication No. WO 86/00912. During surgery one or more pieces of thegel or film, as appropriate, are inserted or injected between or amongthe tissues that are to be kept separate.

The insoluble materials of the invention can also be used as surfacepacification agents, both covalently and/or non-covalently attached tobiodurable and erodible polymer surfaces; as sealing agents inanastomotic sites for catheters, bowel anastomosis, endoscopic surgicalprocedure, vascular grafts, and any prosthetic device requiring gluingtogether or sealing of potential leakage sites; as a potentially newbiocompatible fiber for processing into thread, braids, woven andnon-woven webs, weaves, and mats, and sutures for wound closure;sclerosing agents for varicose vein removal, tumors, and aneurism;artificial extracellular matrix material for tissue replacement in skinlacerations and burns.

Films, foams or gels of the invention can further be used for sustainedrelease drug delivery. The drug to be delivered can be covalently bondedto the gel or film, as described, for example, in R. V. Sparer et al.,1983, Chapter 6, pages 107-119, in T. J. Roseman et al., ControlledRelease Delivery Systems, Marcel Dekker, Inc., New York; and the gel orfilm can then be implanted or injected at the locus where delivery isdesired.

OTHER EMBODIMENTS

Other embodiments are within the following claims. For example, thescale of the reactions may be increased for commercial production of thecompositions of the invention. It will also be well understood by thoseskilled in the art that varying the ratio of polyanionic polysaccharideto activating agent will control the degree of functionalization of thepolyanionic polysaccharide.

We claim:
 1. A method of making a water insoluble biocompatiblecomposition, said method comprising combining, in an aqueous mixture,(a)a polyanionic polysaccharide at a concentration range between 0.005 to0.1M, wherein said polyanionic polysaccharide has a molecular weight inthe range of 9.0×10⁴ to 3.0×10⁶ daltons, (b) at least 1 molar equivalentof a nucleophile per molar equivalent of said polyanionicpolysaccharide, and (c) at least 0.1 molar equivalent of an activatingagent per molar equivalent of said polyanionic polysaccharide, whereinthe reaction is carried out at a pH of 3.5 to 8.0 under conditionssufficient to form said water insoluble biocompatible composition. 2.The method of claim 1 wherein two or more polyanionic polysaccharidesare employed.
 3. The method of claim 1 or 2 wherein said polyanionicpolysaccharides are chosen from the group consisting of carboxymethylcellulose, carboxymethyl amylose, hyaluronic acid,chondroitin-6-sulfate, and dermatin sulfate.
 4. The method of claim 2wherein two of said polyanionic polysaccharides are hyaluronic acid andcarboxymethyl cellulose.
 5. A water insoluble composition preparedaccording to the method of claim 1 or
 2. 6. The composition of claim 5wherein said composition is in the form of a gel.
 7. The composition ofclaim 5 wherein said composition is in the form of fibers.
 8. Thecomposition of claim 5 wherein said composition is in the form of amembrane.
 9. The composition of claim 5 wherein said composition is inthe form of a foam.
 10. The composition of claim 5, further comprising adrug dispersed within said composition.
 11. The composition of claim 10wherein said drug is selected from the group consisting of proteins,growth factors, enzymes, biopolymers, and biologically compatiblesynthetic polymers.
 12. The composition of claim 5 wherein saidpolyanionic polysaccharides are chosen from the group consisting ofcarboxymethyl cellulose, carboxymethyl amylose, hyaluronic acid,chondroitin-6-sulfate, and dermatin sulfate.
 13. The composition ofclaim 5 wherein said polyanionic polysaccharide is hyaluronic acid. 14.The composition of claim 5 wherein said polyanionic polysaccharide iscarboxymethyl cellulose.
 15. The composition of claim 5 wherein saidpolyanionic polysaccharide is carboxymethyl amylose.
 16. The compositionof claim 5 wherein two of said polyanionic polysaccharides arehyaluronic acid and carboxy methyl cellulose.
 17. The composition ofclaim 5 wherein said nucleophile is chosen from the group consisting ofan amino acid amide, a monofunctional amine, an amino acid ester, anamino alcohol, an amino thiol, an amino phenol, an amino catechol, anamino acid, a salt of an amino acid, a peptide, and a protein.
 18. Themethod of claim 1 wherein said polyanionic polysaccharide is hyaluronicacid.
 19. The method of claim 1 wherein said polyanionic polysaccharideis carboxymethyl cellulose.
 20. The method of claim 1 wherein saidpolyanionic polysaccharide is carboxymethyl amylose.
 21. The method ofclaim 1 wherein said nucleophile is selected from the group consistingof an amino acid amide, a monofunctional amine, an amino acid ester, anamino alcohol, an amino thiol, an amino phenol, an amino catechol, anamino acid, a salt of an amino acid, a peptide, and a protein.